Sheng Wenjie, Zhang Chaoyu, Mohiuddin T M, Al-Rawe Marwah, Schmitz Roland, Niebert Marcus, Konrad Lutz, Wagner Steffen, Zeppernick Felix, Meinhold-Heerlein Ivo, Hussain Ahmad Fawzi
Department of Gynecology and Obstetrics, Medical Faculty, Justus-Liebig-University Giessen, Klinikstr. 33, 35392 Giessen, Germany.
Clinic for Gynecology and Obstetrics, University Hospital Brandenburg, Medizinische Hochschule Brandenburg Campus GmbH, Hochstraße 29, 14770 Brandenburg an der Havel, Germany.
Cells. 2025 May 10;14(10):691. doi: 10.3390/cells14100691.
The immunofluorescence assay is widely used for cellular biology and diagnosis applications. Such an antigen-antibody detection system enables the assessment and visualization of the expression and localization of target proteins. In the classical indirect immunofluorescence assay, secondary antibodies are conjugated to fluorophores. However, conventional secondary antibodies have limited applications due to their large size (150 kDa). Moreover, as animal-derived products, secondary antibodies are associated with ethical concerns and batch-to-batch variability. In this study, we developed fluorescence-labeled recombinant nanobodies as secondary antibodies by utilizing previously established anti-mouse and anti-rabbit IgG secondary nanobodies in combination with the self-labeling SNAP-tag. Nanobodies, which are significantly smaller (15 kDa), are capable to detect primary antibodies produced in mice and rabbits. The SNAP-tag (20 kDa) enables site-specific binding of various O-benzylguanine (BG)-modified fluorophores to the recombinant nanobodies. These recombinant nanobodies were produced using mammalian cell expression system, and their specific binding to mouse or rabbit antibodies was validated using flow cytometry and multi-color fluorescence microscopy. The low cost, easy of expression, purification and site-specific conjugation procedures for these anti-mouse and anti-rabbit IgG secondary nanobodies make them an attractive alternative to traditional secondary antibodies for indirect immunofluorescence assays.
免疫荧光测定法广泛应用于细胞生物学和诊断领域。这种抗原-抗体检测系统能够评估和可视化目标蛋白的表达与定位。在经典的间接免疫荧光测定法中，二抗与荧光团偶联。然而，传统二抗由于其较大的尺寸（150 kDa）而应用受限。此外，作为动物源产品，二抗还存在伦理问题和批次间差异。在本研究中，我们通过利用先前建立的抗小鼠和抗兔IgG二抗纳米抗体与自标记SNAP标签相结合，开发了荧光标记的重组纳米抗体作为二抗。纳米抗体显著更小（15 kDa），能够检测小鼠和兔子产生的一抗。SNAP标签（20 kDa）使各种O-苄基鸟嘌呤（BG）修饰的荧光团能够位点特异性地结合到重组纳米抗体上。这些重组纳米抗体是使用哺乳动物细胞表达系统生产的，并通过流式细胞术和多色荧光显微镜验证了它们与小鼠或兔抗体的特异性结合。这些抗小鼠和抗兔IgG二抗纳米抗体成本低、易于表达、纯化且具有位点特异性偶联程序，使其成为间接免疫荧光测定中传统二抗的有吸引力的替代品。
